Abstract: In human breast milk, over 50% energy is in the form of triacylglycerol (TAG), which is dominated by a functional lipid, 1,3-dioleoyl-2-palmitoyl-glycerol (OPO). A number of studies have shown that OPO possesses particular stereospecific arrangement of acyl chains which is essential for the absorption of fatty acids in infant gut. However, most of the commercial OPOs in the domestic market rely on imports currently. The contradiction between price and quality has restricted the wide application of OPO in the infant fomula. Chlamydomonas reinhardtii is a model organism for investigating lipid metabolism, and numerous studies suggested that C. reinhardtii is able to accumulate OPO under stress conditions such as nitrogen deficiency. This study established a method to quantify 1,3-dioleoyl-2-palmitoyl-glycerol OPO by employing ultra-high performance liquid chromatography coupled with tandem triple quadrupole mass spectrometry. The OPO content was quantified with the internal standard method through multi-reaction monitoring mode (MRM) under positive mode, and the selected ion-pair was 876>577. The OPO production was investigated in the wild-type C. reinhardtii strain cc-5325 and the galactosyl hydrolase gene mutant strain M08. The results showed that the content of OPO in C. reinhardtii accumulated greatly under nitrogen deprivation with great difference in the two strains. Compared to cc-5325, from day1 to day3, nitrogen deprivation increased the content of OPO of M08 by 3.70, 3.04 and 2.74 times, and it increased the yield of OPO of M08 by 1.13, 1.53 and 1.33 times, respectively. These data suggest that modifying lipid metabolic pathway can increase the production of OPO in C. reinhardtii. Therefore, C. reinhardtii has potential as a new food ingredient for commercial oils. This study provide technical support and basis to increase OPO production in different genetically engineered C. reinhardtii.